Human gene transfer involves the transfer of one or more therapeutic genes and the sequences controlling their expression to appropriate target cells. A number of vector systems have been developed for the transfer of the therapeutic genes for various clinical indications. In vivo gene transfer involves the direct administration of vector to the target cells within a patient. Ex vivo gene transfer entails removing target cells from an individual, modifying them ex vivo and returning the modified cells to the patient.
The majority of gene therapy protocols approved for clinical trials by the NIH Recombinant DNA Advisory Committee (RAC) have used arnphotropic retroviral vectors (ORDA Reports Recombinant DNA Advisory Committee (RAC) Data Management Report, June 1994, (1994) Human Gene Therapy 5:1295-1302). Retroviral vectors are the vehicle of choice primarily due to the generally high rate of gene transfer obtained in experiments with cell lines and the ability to obtain stable integration of the genetic material, ensuring that the progeny of the modified cell will contain the transferred genetic material. For a review of retroviral vectors and their use in the transfer and expression of foreign genes, see Gilboa (1988) Adv. Exp. Med. Biol. 241:29; Luskey et al. (1990) Ann. N.Y. Acad. Sci. 612:398; and Smith (1992) J. Hematother. 1:155-166.
Many retroviral vectors currently in use are derived from the Moloney murine leukemia virus (MMLV). In most cases, the viral gag, pol and env sequences are removed from the virus, allowing for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are often expressed under the control of the strong viral promoter in the LTR. Such a construct can be packaged into vector particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the Gag-Pol and Env proteins produced by the cell assemble with the vector RNA to produce replication-defective or transducing virions that are secreted into the culture medium. The vector particles thus produced can infect and integrate into the DNA of the target cell, but generally will not produce infectious virus since it is lacking essential viral sequences.
Most of the packaging cell lines currently in use have been transfected with separate plasmids encoding Gag-Pol and Env, so that multiple recombination events are necessary before a replication-competent retrovirus (RCR) can be produced. Commonly used retroviral vector packaging cell lines are based on the murine NIH/3T3 cell line and include PA317 (Miller & Buttimore (1986) Mol. Cell Biol. 6:2895; Miller & Rosman (1989) BioTechniques 7:980), CRIP (Danos & Mulligan (1988) Proc. Natl Acad Sci USA 85:6460), and gp+am12 (Markowitz et al. (1988) Virology 167:400). Although splitting the gag-pol and env genes within the packaging cell genome decreases the incidence of RCR, RCR is occasionally observed in clinical-scale productions of retroviral vector preparations and is a major safety concern. This is likely due, at least in part, to the fact that NIH/3T3 cells contain endogenous MLV sequences (Irving et al. (1993) Bio/Technol. 11:1042-1046) which could participate in recombination to form RCR (Cosset et al. (1993) Virology 193:385-395 and Vanin et al. (1994) J Virology 68:4241-4250), particularly in mass culture during large-scale clinical vector production.
The range of host cells that may be infected by a retrovirus or transduced by a retroviral vector is determined by the viral Env protein. The recombinant virus can be used to infect virtually any cell type recognized by the Env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product. The efficiency of infection is also related to the level of expression of the receptor on the target cell. In general, murine ecotropic Env of MMLV allows infection of rodent cells, whereas amphotropic Env allows infection of rodent, avian and some primate cells, including human cells. Xenotropic vector systems utilizing murine xenotropic Env would also allow transduction of human cells.
The host range of retroviral vectors has been altered by substituting the Env protein of the base virus with that of a second virus. The resulting, "pseudotyped" vector particle has the host range of the virus donating the envelope protein and expressed by the packaging cell line. For example, the G-glycoprotein from vesicular stomatitis virus (VSV-G) has been substituted for the MMLV Env protein, thereby broadening the host range. See, e.g., Burns et al. (1993) Proc. Natl. Acad. Sci USA 90:8033-8037 and International PCT patent application Publication No. WO 92/14829.
Inconsistent results and inefficient gene transfer to some target cell types are two additional problems associated with current retroviral vector systems. For example, hematopoietic stem cells are an attractive target cell type for gene therapy because of their self-renewal capacity and their ability to differentiate into all hematopoietic lineages, thereby repopulating a patient with the modified cells. Yet retroviral gene transfer into hematopoietic stem cells has been inconsistent and disappointingly inefficient. Kantoff et al. (1987) J. Exp. Med. 166:219-234; Miller, A. D. (1990) Blood 76:271-278; and Xu et al. (1994) J. Virol. 68:7634. Efforts to increase gene transfer efficiency include producing higher end-point-titer retroviral vector supernatants. End-point titer is a measure of the number of functional vector particles in a preparation which, when increased, should theoretically increase transduction efficiency by increasing the ratio of functional vector to target cells, i.e. increasing multiplicity of infection (m.o.i.). Despite increased end-point titers, however, retroviral gene transfer efficiency (transduction efficiency) has not increased correspondingly (Xu et al. (1994), supra; Paul (1993) Hum. Gene Therapy 4:609-615; Fraes-Lutz et al. (1994) 22:857-865).
Efforts to increase end-point titer have included improving production of retroviral vector supernatants (see Kotani et al. (1994) Human Gene Therapy, 5:19-28) and physical concentration of vector particles by ultrafiltration (Paul, et al. (1993), supra. and Kotani, et al. (1994) supra). It was shown that incubation of producer cells at 32.degree. C. rather than at 37.degree. C. yielded supernatants with higher end-point titers, but transduction efficiencies were not compared (See Kotani, et al. (1994) supra). The authors of Kotani et al. (1994) supra, postulated that the higher titers were due to a lower rate of inactivation combined with a faster rate of virion production at 32.degree. C. In another study, transduction efficiency was measured before and after concentration of three supernatants with similar end-point titers Paul et al. (1993) supra. In each case, concentration increased end-point titer and modestly improved the transduction efficiency. However, the transduction efficiency achieved with one of the unconcentrated supernatants was significantly higher than that achieved with the other concentrates (Paul et al. (1993) supra).
For in vivo gene therapy applications, it is important that the retroviral vector not be inactivated by human serum before transducing the target cells. Reports show that human serum inactivates a number of recombinant retroviruses, apparently via a complement pathway. Both viral envelope and producer cell components have been reported to be responsible for viral sensitivity to human complement (Takeuchi et al. (1994) J. Virol. 68(12):8001).
Thus, a need exists for methods of reproducibly increasing transduction efficiency and for providing stable, safe packaging cell lines for producing high transduction efficiency retroviral preparations. This invention satisfies these needs and provides related advantages as well.